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1.
Viruses ; 15(1)2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36680287

RESUMO

The transmembrane protein Axl was proposed as an entry receptor for Zika virus (ZIKV) infection in vitro, but conflicting results from in vivo studies have made it difficult to establish Axl as a physiologically relevant ZIKV receptor. Both the functional redundancy of receptors and the experimental model used can lead to variable results. Therefore, it can be informative to explore alternative animal models to analyze ZIKV receptor candidates as an aid in discovering antivirals. This study used chicken embryos to examine the role of chicken Tyro3 (cTyro3), the equivalent of human Axl. Results show that endogenous cTyro3 mRNA expression overlaps with previously described hot spots of ZIKV infectivity in the brain and inner ear. We asked if ectopic expression or knockdown of cTyro3 influenced ZIKV infection in embryos. Tol2 vectors or replication-competent avian retroviruses were used in ovo to introduce full-length or truncated (presumed dominant-negative) cTyro3, respectively, into the neural tube on embryonic day two (E2). ZIKV was delivered to the brain 24 h later. cTyro3 manipulations did not alter ZIKV infection or cell death in the E5/E6 brain. Moreover, delivery of truncated cTyro3 variants to the E3 otocyst had no effect on inner ear formation on E6 or E10.


Assuntos
Orelha Interna , Infecção por Zika virus , Zika virus , Embrião de Galinha , Animais , Humanos , Receptores Proteína Tirosina Quinases/metabolismo , Galinhas , Proteínas Proto-Oncogênicas/metabolismo , Receptores Virais/metabolismo , Encéfalo/metabolismo , Replicação Viral
2.
Opt Lett ; 48(2): 379-382, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36638462

RESUMO

We propose an absorption intensity heartbeat modulation-averaged shifted histogram (AIHM-ASH) method for estimating human heart rate (HR) using color videos of lip image sequences. When heartbeat occurs, AIHM is generated. Based on the AIHM, HR signals can be demodulated by computing the instantaneous HR modulation depth that presents the relative red blood cell (RBC) concentration from the green channel image of the RGB color video. In addition, the ASH algorithm further suppresses the background tissue and vein signals, and increases the signal-to-noise ratio (SNR). The experimental results for flow phantoms, chicken embryos, and human lips validated the proposed method's optimal estimation conditions and effectiveness, where the accuracy and root mean square error (RMSE) were 99.23% and 0.8 bpm, respectively. The proposed HR estimation method has significant potential to advance health monitoring and disease prevention via conventional color video cameras installed in public places.


Assuntos
Algoritmos , Embrião de Galinha , Humanos , Animais , Frequência Cardíaca/fisiologia , Razão Sinal-Ruído , Cor
3.
Epigenetics Chromatin ; 16(1): 2, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36617567

RESUMO

BACKGROUND: Sex determination and differentiation are complex and delicate processes. In female chickens, the process of sex differentiation is sensitive and prone to be affected by the administration of aromatase inhibitors, which result in chicken sex reversal and infertility. However, the molecular mechanisms underlying sex differentiation and infertility in chicken sex reversal remain unclear. Therefore, we established a sex-reversed chicken flock by injecting an aromatase inhibitor, fadrozole, and constructed relatively high-resolution profiles of the gene expression and chromatin accessibility of embryonic gonads. RESULTS: We revealed that fadrozole affected the transcriptional activities of several genes, such as DMRT1, SOX9, FOXL2, and CYP19A1, related to sex determination and differentiation, and the expression of a set of gonadal development-related genes, such as FGFR3 and TOX3, by regulating nearby open chromatin regions in sex-reversed chicken embryos. After sexual maturity, the sex-reversed chickens were confirmed to be infertile, and the possible causes of this infertility were further investigated. We found that the structure of the gonads and sperm were greatly deformed, and we identified several promising genes related to spermatogenesis and infertility, such as SPEF2, DNAI1, and TACR3, through RNA-seq. CONCLUSIONS: This study provides clear insights into the exploration of potential molecular basis underlying sex differentiation and infertility in sex-reversed chickens and lays a foundation for further research into the sex development of birds.


Assuntos
Galinhas , Infertilidade , Embrião de Galinha , Animais , Feminino , Masculino , Galinhas/genética , Diferenciação Sexual/genética , Fadrozol/metabolismo , Fadrozol/farmacologia , Sequenciamento de Cromatina por Imunoprecipitação , RNA-Seq , Processos de Determinação Sexual , Sêmen , Gônadas/metabolismo , Inibidores da Aromatase/metabolismo , Cromatina/genética , Cromatina/metabolismo , Infertilidade/genética , Infertilidade/metabolismo , Regulação da Expressão Gênica no Desenvolvimento
4.
Sci Rep ; 13(1): 263, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36609620

RESUMO

Neural tube closure (NTC) is a complex process of embryonic development involving molecular, cellular, and biomechanical mechanisms. While the genetic factors and biochemical signaling have been extensively investigated, the role of tissue biomechanics remains mostly unexplored due to the lack of tools. Here, we developed an optical modality that can conduct time-lapse mechanical imaging of neural plate tissue as the embryo is experiencing neurulation. This technique is based on the combination of a confocal Brillouin microscope and a modified ex ovo culturing of chick embryo with an on-stage incubator. With this technique, for the first time, we captured the mechanical evolution of the neural plate tissue with live embryos. Specifically, we observed the continuous increase in tissue modulus of the neural plate during NTC for ex ovo cultured embryos, which is consistent with the data of in ovo culture as well as previous studies. Beyond that, we found that the increase in tissue modulus was highly correlated with the tissue thickening and bending. We foresee this non-contact and label-free technique opening new opportunities to understand the biomechanical mechanisms in embryonic development.


Assuntos
Microscopia , Neurulação , Animais , Feminino , Gravidez , Embrião de Galinha , Microscopia/métodos , Tubo Neural , Imagem com Lapso de Tempo , Desenvolvimento Embrionário
5.
Toxicology ; 485: 153414, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36587891

RESUMO

Human exposure to plastic contaminated foods and environmental micro/nano plastic derived chemicals necessitates system-wide health risk assessment. Hence, current study intend to explore the mode of action (MoA) based adverse outcome pathways of 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), the major active metabolite of bisphenol A (BPA). The computational study employed broad range of target prediction, systems biology tools and molecular docking protocols. Further, validation of MBP targets was done using protein-ligand fluorescence quenching assay, endothelial cell culture and chicken embryo vascular angiogenesis models. Interestingly, the current results illustrate that various physiological signaling pathways (MAPK and VEGF related angiogenesis signaling) and disease progression pathways (hypertension, cancer and endocrine disorders) were enriched as potential targets of MBP. Further, docking studies highlights the possible binding mechanism of MBP with important targets including endothelial nitric oxide synthase (eNOS) and serum albumin (BSA). In addition, the validation studies on MBP-BSA interaction (fluorescence quenching), eNOS derived nitric oxide (NOx) generation in endothelial cells and chicken embryo angiogenesis support the system-wide impacts of MBP with highlights on cardiovascular pathogenesis. Thus, the current observation provides novel insights into the system wide impacts of MBP for the futuristic health risk assessment of plastic derived chemicals.


Assuntos
Poluentes Ambientais , Animais , Humanos , Embrião de Galinha , Simulação de Acoplamento Molecular , Células Endoteliais/metabolismo , Compostos Benzidrílicos/química
6.
Biophys Chem ; 292: 106913, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36330890

RESUMO

Curcumin has attracted more attention because of its inhibition efficacy on protein amyloid fibrillation. However, the inhibition mechanism was still ambiguous and the clinical application of curcumin was greatly limited because of its poor stability at physiological conditions for the presence of ß-diketone moiety. In this paper, a new mono-ketone-containing curcumin analogue (MDHC) was designed and synthesized to realize the possible inhibition mechanism and unveil the important role of ß-diketone moiety of curcumin in the inhibition process of amyloid fibrillation using hen egg white lysozyme (HEWL) as model protein. Although all experiment results (ThT, CR, ANS and TEM) showed that the inhibitory capacity of curcumin was better than MDHC, MDHC still could show obvious inhibition effect. Molecular docking showed that both curcumin and MDHC could bind with HEWL by hydrogen bond of phenloic hydroxyl and the binding energy of MDHC was higher than that of curcumin. All the findings inferred that ß-diketone group was one of great important groups in the inhibition process of HEWL amyloid fibrillation, which provided more room to construct novel inhibition reagents.


Assuntos
Amiloidose , Curcumina , Amiloide/química , Proteínas Amiloidogênicas , Clara de Ovo , Simulação de Acoplamento Molecular , Muramidase/química , Animais , Embrião de Galinha
7.
Artigo em Inglês | MEDLINE | ID: mdl-36244467

RESUMO

Embryonic morphine exposure (EME) leads to abnormal brain development and behavior in the offspring, and the functional alteration of γ-aminobutyric acid (GABA) system is considered to be one of the important mechanisms. To mimic the problem of susceptibility of human gestational drug abuse on addictive drugs in offspring, we administered morphine exposure on days 5-8 and 13-16 of chicken embryo development and examined the functions of GABA neurons and their receptors in postnatal chicks by neuroelectrophysiology, immunohistochemistry and behavioral methods. We found that morphine exposure during embryonic stages 5-8 (MorphineE5-8) significantly reduced the incidence of spontaneous inhibitory postsynaptic potentiation (IPSP) and the induction of evoked IPSP and the mean amplitude of GABAA agonist muscimol-induced response in the intermediate medial interstitial (IMM) region, compared to naïve controls or saline-exposed chicks. The results of immunocytochemistry further suggest that MorphineE5-8 decreased the synaptic density of GAD-expressing sites in the IMM, while increased the expression of the GABAA receptor subtype γ2 isoform. Behavioral results found that Morphine5-8 treatment de-inhibited morphine-induced psychomotor responses in postnatal chicks. Morphine exposure at embryonic stages 13-16 (MorphineE13-16) showed no significant changes in the above indicators compared to the saline group. Evidence suggests that early embryonic morphine exposure leads to defects in GABAergic function in the IMM, which in turn alters the responsiveness of postnatal chicks to addictive drugs. These results will help to understand the GABA mechanisms by which embryonic addictive drug exposure contributes to offspring susceptibility to addiction.


Assuntos
Galinhas , Morfina , Humanos , Animais , Embrião de Galinha , Morfina/farmacologia , Galinhas/metabolismo , Ácido gama-Aminobutírico/metabolismo , Encéfalo/metabolismo , Neurônios , Receptores de GABA-A/metabolismo
8.
Res Vet Sci ; 154: 73-77, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36495713

RESUMO

Folate plays important role in biosynthesis of purine and pyrimidine nucleotides and is therefore crucial for DNA synthesis and neurogenesis in fetal brains. Many genes comprising brain derived neurotrophic factor (BDNF) and miRNAs have been shown to play important role in brain development. Gga-miR-190a-3p targets many genes including BDNF. The aim of this project was to study the effects of in ovo administration of folic acid (FA) on BDNF and gga-miR-190a-3p expression in the cerebral cortex of chick embryo. A total number of 120 hatching eggs with the correct shape and weight were used in this experiment. Forty eggs was injected by FA into the yolk sac at a dose of 150-µg per egg, 40 eggs by PBS (SHAM) on embryonic day 11 and 40 eggs were left without injection as controls. Then the cerebral cortex was collected on E19 and BDNF and gga-miR-190a-3p expression was studied using Real time PCR. The results showed that BDNF expression in the cortex of FA treated, SHAM and controls were 2.06 ± 0.29, 1.12 ± 0.12 and 1.02 ± 0.21 fold changes, respectively and for gga-miR-190a-3p were 0.72 ± 0.08, 0.95 ± 0.09 and 1.007 ± 0.12 fold change, respectively. Statistical analysis showed that there is significant increase in BDNF expression and decreased gga-miR-190a-3p expression in FA injected cerebral cortex as compared either with SHAM or controls. Although, no significant change in BDNF and gga-miR-190a-3p expression were observed between SHAM and controls. It is concluded that in ovo administration of FA increases BDNF and decreases gga-miR-190a-3p expression in the developing chick cerebral cortex.


Assuntos
Galinhas , MicroRNAs , Embrião de Galinha , Animais , Galinhas/genética , Fator Neurotrófico Derivado do Encéfalo/genética , Ácido Fólico/farmacologia , Óvulo/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo
9.
Vet Microbiol ; 277: 109619, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36525909

RESUMO

The virulence of avian gamma-coronavirus infectious bronchitis viruses (IBV) for the kidney has led to high mortality in dominant-genotype isolations, but the key sites of viral protein that determine kidney tropism are still not fully clear. In this study, the amino acid sequences of the S2 subunit of IBVs with opposing adaptivity to chicken embryonic kidney cells (CEKs) were aligned to identify putative sites associated with differences in viral adaptability. The S2 gene and the putative sites of the non-adapted CN strain were introduced into the CEKs-adapted SczyC30 strain to rescue seven mutants. Analysis of growth characteristics showed that the replacement of the entire S2 subunit and the L1089I substitution in the S2 subunit entirely abolished the proliferation of recombinant IBV in CEKs as well as in primary chicken oviduct epithelial cells. Pathogenicity assays also support the decisive role of this L1089 for viral nephrotropism, and this non-nephrotropic L1089I substitution significantly attenuates pathogenicity. Analysis of the putative cause of proliferation inhibition in CEKs suggests that the L1089I substitution affects neither virus attachment nor endocytosis, but instead fails to form double-membrane vesicles to initiate the viral replication and translation. Position 1089 of the IBV S2 subunit is conservative and predicted to lie in heptad repeat 2 domains. It is therefore reasonable to conclude that the L1089I substitution alters the nephrotropism of parent strain by affecting virus-cell fusion. These findings provide crucial insights into the adaptive mechanisms of IBV and have applications in the development of vaccines and drugs against IB.


Assuntos
Infecções por Coronavirus , Vírus da Bronquite Infecciosa , Doenças das Aves Domésticas , Embrião de Galinha , Animais , Fusão Celular/veterinária , Galinhas , Tropismo Viral , Rim , Tropismo , Infecções por Coronavirus/veterinária , Glicoproteína da Espícula de Coronavírus/genética
10.
Vet Microbiol ; 277: 109625, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36563582

RESUMO

In ovo vaccination is an attractive immunization approach for the poultry industry. However, commonly used Newcastle disease virus (NDV) vaccines cannot be administered in ovo because of the reduced hatchability and embryo mortality. The codon pair deoptimization (CPD) approach has been used to efficiently and rapidly attenuate viruses by targeting the virulence genes. In this study, we aimed to attenuate the NDV LaSota (LS) vaccine strain for in ovo vaccination by CPD of the fusion (F) or/and hemagglutinin-neuraminidase (HN) genes with approximately 44 % suboptimal codon substitutions. Three NDV LS recombinants expressing codon deoptimized F (rLS/F-d), HN (rLS/HN-d), or both genes (rLS/F+HN-d) were generated using reverse genetics technology. Biological assays showed that the CPD viruses retained similar hemagglutination activity and growth ability to the parental rLS virus. The CPD of the HN gene slightly attenuated the rLS/HN-d and rLS/F+HN-d viruses, whereas the CPD of the F gene marginally increased the rLS/F-d virus pathogenicity compared to rLS. Nevertheless, all three CPD rLS viruses were still lethal to 10-day-old specific-pathogen-free (SPF) chicken embryos. In ovo inoculation of 18-day-old SPF chicken embryos with the CPD viruses severely reduced chicken's hatch and survival rates. These results suggested that the CPD of the surface glycoprotein genes of the LS strain at the current level of suboptimal codon substitutions could not sufficiently attenuate the virus for use as an in ovo vaccine, and codon deoptimizing a greater proportion of the F and HN genes or additional gene(s) may be required for sufficient attenuation of the LS strain.


Assuntos
Doença de Newcastle , Vacinas Virais , Embrião de Galinha , Animais , Vírus da Doença de Newcastle , Doença de Newcastle/prevenção & controle , Galinhas , Vacinação/veterinária , Vacinação/métodos , Proteína HN/genética , Códon
11.
BMC Genomics ; 23(1): 825, 2022 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-36513979

RESUMO

BACKGROUND: The transition from fertilized egg to embryo in chicken requires activation of hundreds of genes that were mostly inactivated before fertilization, which is accompanied with various biological processes. Undoubtedly, transcription factors (TFs) play important roles in regulating the changes in gene expression pattern observed at early development. However, the contribution of TFs during early embryo development of chicken still remains largely unknown that need to be investigated. Therefore, an understanding of the development of vertebrates would be greatly facilitated by study of the dynamic changes in transcription factors during early chicken embryo. RESULTS: In the current study, we selected five early developmental stages in White Leghorn chicken, gallus gallus, for transcriptome analysis, cover 17,478 genes with about 807 million clean reads of RNA-sequencing. We have compared global gene expression patterns of consecutive stages and noted the differences. Comparative analysis of differentially expressed TFs (FDR < 0.05) profiles between neighboring developmental timepoints revealed significantly enriched biological categories associated with differentiation, development and morphogenesis. We also found that Zf-C2H2, Homeobox and bHLH were three dominant transcription factor families that appeared in early embryogenesis. More importantly, a TFs co-expression network was constructed and 16 critical TFs were identified. CONCLUSION: Our findings provide a comprehensive regulatory framework of TFs in chicken early embryo, revealing new insights into alterations of chicken embryonic TF expression and broadening better understanding of TF function in chicken embryogenesis.


Assuntos
Galinhas , Fatores de Transcrição , Embrião de Galinha , Animais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Galinhas/genética , Galinhas/metabolismo , Transcriptoma , Perfilação da Expressão Gênica , Desenvolvimento Embrionário/genética
12.
Int J Mol Sci ; 23(23)2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36499456

RESUMO

Matrix vesicles (MVs) contain the whole machinery necessary to initiate apatite formation in their lumen. We suspected that, in addition to tissue-nonspecific alkaline phosphatase (TNAP), Na,K,-ATPase (NKA) could be involved in supplying phopshate (Pi) in the early stages of MV-mediated mineralization. MVs were extracted from the growth plate cartilage of chicken embryos. Their average mean diameters were determined by Dynamic Light Scattering (DLS) (212 ± 19 nm) and by Atomic Force Microcopy (AFM) (180 ± 85 nm). The MVs had a specific activity for TNAP of 9.2 ± 4.6 U·mg-1 confirming that the MVs were mineralization competent. The ability to hydrolyze ATP was assayed by a colorimetric method and by 31P NMR with and without Levamisole and SBI-425 (two TNAP inhibitors), ouabain (an NKA inhibitor), and ARL-67156 (an NTPDase1, NTPDase3 and Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) competitive inhibitor). The mineralization profile served to monitor the formation of precipitated calcium phosphate complexes, while IR spectroscopy allowed the identification of apatite. Proteoliposomes containing NKA with either dipalmitoylphosphatidylcholine (DPPC) or a mixture of 1:1 of DPPC and dipalmitoylphosphatidylethanolamine (DPPE) served to verify if the proteoliposomes were able to initiate mineral formation. Around 69-72% of the total ATP hydrolysis by MVs was inhibited by 5 mM Levamisole, which indicated that TNAP was the main enzyme hydrolyzing ATP. The addition of 0.1 mM of ARL-67156 inhibited 8-13.7% of the total ATP hydrolysis in MVs, suggesting that NTPDase1, NTPDase3, and/or NPP1 could also participate in ATP hydrolysis. Ouabain (3 mM) inhibited 3-8% of the total ATP hydrolysis by MVs, suggesting that NKA contributed only a small percentage of the total ATP hydrolysis. MVs induced mineralization via ATP hydrolysis that was significantly inhibited by Levamisole and also by cleaving TNAP from MVs, confirming that TNAP is the main enzyme hydrolyzing this substrate, while the addition of either ARL-6715 or ouabain had a lesser effect on mineralization. DPPC:DPPE (1:1)-NKA liposome in the presence of a nucleator (PS-CPLX) was more efficient in mineralizing compared with a DPPC-NKA liposome due to a better orientation of the NKA active site. Both types of proteoliposomes were able to induce apatite formation, as evidenced by the presence of the 1040 cm-1 band. Taken together, the findings indicated that the hydrolysis of ATP was dominated by TNAP and other phosphatases present in MVs, while only 3-8% of the total hydrolysis of ATP could be attributed to NKA. It was hypothesized that the loss of Na/K asymmetry in MVs could be caused by a complete depletion of ATP inside MVs, impairing the maintenance of symmetry by NKA. Our study carried out on NKA-liposomes confirmed that NKA could contribute to mineral formation inside MVs, which might complement the known action of PHOSPHO1 in the MV lumen.


Assuntos
Calcinose , Monoéster Fosfórico Hidrolases , Animais , Embrião de Galinha , Monoéster Fosfórico Hidrolases/metabolismo , ATPase Trocadora de Sódio-Potássio , Calcificação Fisiológica , Fosfatase Alcalina/metabolismo , Hidrólise , Trifosfato de Adenosina , Lipossomos/química , Minerais/metabolismo
13.
Molecules ; 27(24)2022 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-36558133

RESUMO

Transition metal complexes have continued to constitute an appealing class of medicinal compounds since the exceptional discovery of cisplatin in the late 1960s. Pt(II)-based complexes are endowed with a broad range of biological properties, which are mainly exerted by targeting DNA. In this study, we report a significant biological investigation into and computation analyses of four Pt(II)-complexes, namely, LDP-1-4, synthesized and characterized according to previously reported procedures. Molecular-modelling studies highlighted that the top two LDP compounds (i.e., LDP-1 and LDP-4) might bind to both matched and mismatched base pair sites of the oligonucleotide 5'-(dCGGAAATTACCG)2-3', supporting their anticancer potential. These two complexes displayed noteworthy cytotoxicity in vitro (sub-micromolar-micromolar range) against two leukaemia cell lines, i.e., CCRF-CEM and its multi-drug-resistant counterpart CEM/ADR5000, and remarkable anti-angiogenic properties (in the sub-micromolar range) evaluated in an in vivo model, i.e., a chick embryo chorioallantoic membrane (CAM) assay.


Assuntos
Antineoplásicos , Complexos de Coordenação , Animais , Embrião de Galinha , Platina/farmacologia , Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Cisplatino , DNA , Linhagem Celular Tumoral
14.
Adv Exp Med Biol ; 1383: 105-111, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36587150

RESUMO

I outline here the development of intestinal motility in the chicken embryo. The first contractile events are circular smooth muscle driven calcium waves (E6), that gain a clock-like regularity when interstitial cells of Cajal become electrically active (E14). Soon after longitudinal smooth muscle contractions become prominent (E14), the enteric nervous system starts controlling motility (E16) by coupling the longitudinal and circular contractions via inhibitory neurotransmission. It gives rise to circular-longitudinal antagonism, to the migrating motor complex, and to the polarized ascending contraction-descending relaxation pressure response known as the "law of the intestine". The kinetics of gut development in the chicken appears to follow faithfully that of humans by simply converting embryonic days of chicken development into embryonic weeks of human development.


Assuntos
Galinhas , Células Intersticiais de Cajal , Animais , Embrião de Galinha , Humanos , Intestinos/fisiologia , Motilidade Gastrointestinal/fisiologia , Células Intersticiais de Cajal/fisiologia , Contração Muscular/fisiologia , Desenvolvimento Embrionário
15.
Klin Lab Diagn ; 67(11): 668-671, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36398777

RESUMO

Using the example of the clinical strain of R. sibirica «Bayevo 105/87¼, the possibility of quantitative determination of rickettsias in clinical samples from patients with Siberian tick-borne typhus by real-time polymerase chain reaction (PCR-RT) was evaluated. Cultivation was carried out in the yolk sacs of developing chicken embryos, from which a piece of the yolk sac or chorion was taken. A total of 125 samples were examined. A set of reagents "RealBest DNA Rickettsia species (kit1)" was used for PCR-RT. The obtained values of the threshold amplification cycle (Ct) were compared with the results of microscopy of smear preparations stained by the Zdrodovsky method, the values of which were divided into ranks: the I rank - single rickettsias in individual fields of vision, the II rank - single rickettsias in each field of vision, the III rank - from 10 to 25 rickettsias in each field of vision, the IV rank - from 25 to 50 rickettsias in each field of view. The median Ct value for rank I was 17.6 (16.37; 18.58), for the II - 16.0 (15.0; 16.41), for the III - 15.0 (14.0; 15.1) and for the IV - 15.0 (13.7; 14.64). A significant average correlation was established between the number of rickettsias in the preparation under microscopy and the value of the threshold cycle in PCR RT (r=-0, 4849542; p=9.968e-09). When determining the correlation between the pathomorphological characteristic and the value of the threshold cycle, its absence was established. The detection of rickettsias in the blood vessels of the chorion of developing chicken embryos was of interest. In 10 samples, the yolk sac and chorion were taken for the study, and in parallel they were examined by PCR-RT. The use of modern, more sensitive molecular biological methods allows for quantitative analysis of DNA in the chorion, while preserving the volumes of the most valuable material - the yolk sac.


Assuntos
Infecções por Rickettsia , Rickettsia , Doenças Transmitidas por Carrapatos , Animais , Embrião de Galinha , Reação em Cadeia da Polimerase em Tempo Real/métodos , Rickettsia/genética , Infecções por Rickettsia/diagnóstico
16.
Arch Microbiol ; 204(12): 715, 2022 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-36400871

RESUMO

Bacillus subtilis (BS) has been used as an excellent probiotic; however, some BS strains seem to be opportunist pathogens or do not present inhibitory effects in the pathogenic bacteria, so the characterization of BS strains for use in animals is mandatory. This study aimed to select nonpathogenic strains of BS, which can inhibit Salmonella spp., avian pathogenic Escherichia coli (APEC), and Campylobacter jejuni (CJ) using a chicken embryo as a model. We tested nine (9) strains of BS isolated from several sources (named A to I) in in vitro by tests of mucin degradation activity, haemolytic activity, apoptosis, and necrosis in fibroblasts from chickens. After the in vitro test, we tested the remaining seven (7) strains (strains A to G) in a chicken embryo (CE) as an in vivo model and target animal. We inoculated 3 log CFU/CE of each strain via allantoic fluid at the 10th day postincubation (DPI). Each treatment group consisted of eight CEs. At the 17th DPI we checked CE mortality, gross lesions, CE weight, and whether BS strains were still viable. To perform the cytokine, total protein, albumin, and reactive C protein analysis, we collected the CE blood from the allantoic vessel and intestine fragments in the duodenum portion for histomorphometric analysis. After the results in CEs, we tested the inhibition capacity of the selected BS strains for diverse strains of Salmonella  Heidelberg (SH), S. Typhimurium (ST), S. Enteritidis (SE), S. Minnesota (SM), S. Infantis (SI), Salmonella var. monophasic (SVM), APEC and C. jejuni. After the in vitro trial (mucin degradation activity, haemolytic activity, apoptosis, and necrosis), we removed two (2) strains (H and I) that showed ß-haemolysis, mucin degradation, and/or high apoptosis and necrosis effects. Although all strains of BS were viable in CEs at the 17th DPI, we removed four (4) strains (A, B, D, F) once they led to the highest mortality in CEs or a high albumin/protein ratio. C. jejuni inoculated with strain G had greater weight than the commercial strain, which could be further used for egg inoculation with benefits to the CE. From the tests in CEs, we selected the strains C, E, and G for their ability to inhibit pathogenic strains of relevant foodborne pathogens. We found that the inhibition effect was strain dependent. In general, strains E and/or G presented better or similar results than commercial control strains in the inhibition of SH, ST, SI, APEC, and two (2) strains of CJ. In this study, we selected BS strains C, E and G due to their in vitro and in vivo safety and beneficial effects. In addition, we emphasize the value of CE as an in vivo experimental model for assessing BS's safety and possible benefits for poultry and other animals.


Assuntos
Campylobacter jejuni , Infecções por Escherichia coli , Probióticos , Embrião de Galinha , Animais , Galinhas/microbiologia , Bacillus subtilis , Escherichia coli , Mucinas , Necrose
17.
Eur Phys J E Soft Matter ; 45(11): 93, 2022 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-36401057

RESUMO

The formation of sensory organs is an important developmental and evolutionary question. In the context of regenerative medicine also, it is important to know as accurately as possible how sensory organs form. The formation of ears, eyes or nose stems presumably from tissue thickenings called placodes Graham and Shimeld (J Anat 222(1):32-40, 2013), Horie et al. (Nature 560:228-232, 2018) which become these organs after processes termed inductions. However, the origin of the placodes, the mechanism of induction and the overall face organization are not understood. Recently, it has been suggested that there is a physical principle to face organization. Indeed, it has been shown that there exists a pattern of rings and rays in the early blastula which defines the position of face landmarks, especially the ears and eyes Fleury et al. (Second order division in sectors as a prepattern for sensory organs in vertebrate development, 2021), Fleury and Abourachid (Eu Phys J E 45:31, 2022). Tensions in the sectors defined by the intersections of the said rings and rays create the actual face features. I report here that a similar situation exists for the nose. This explains the robustness of face formation in the chordates phylum. By studying nasal pit formation in the chicken embryo by time-lapse (T-L) video microscopy, I show that the nasal placode originates in a narrow sector deformed by tension forces following the biaxial pattern of rings and rays mentioned above. Cells align in the pattern and exert organized forces. Further contractions of the pattern contribute to inducing the nasal pit. The observation of the early pre-pattern of lines which locks the facial features explains readily a number of facts regarding sensory organs. Especially the existence of a lacrimal canal between the eye and the nose Lefevre and Freitag (Semin Ophthalmo l 27(5-6):175-86, 2012), or of a slit connecting the nose to the mouth, the correlation between nose, mouth and eye morphogenesis Dubourg et al. (J Rare Dis 2(8), 2007), the presence of shallow valleys on the nasal and optic vesicles, the medio-lateral asymmetry of nostrils with often a bent slit Liu et al. (PLoS ONE 12: e0181928, 2017), the uneven number of nostrils in many fish Cox (J R Soc Interf 5(23):575-593, 2008) and possibly the transition between agnatha and gnathostomes Gai and Zhu (Chinese Sci Bull 57(31), 2012): all appear under this light, geometrically straightforward. The nasal pit forms in a sector of tissue which was present on the blastodic (early embryonic stage), and which is projected onto the nasal vesicle during neurulation. The nasal pit forms along a hairpin of tissue. The top part of the hairpin forms the nares, and the bottom part a groove often visible in many animals.


Assuntos
Nariz , Embrião de Galinha , Animais , Masculino , Bovinos , Morfogênese
18.
PLoS Biol ; 20(11): e3001853, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36395107

RESUMO

The accurate construction of neural circuits requires the precise control of axon growth and guidance, which is regulated by multiple growth and guidance cues during early nervous system development. It is generally thought that the growth and guidance cues that control the major steps of axon development have been defined. Here, we describe cerebellin-1 (Cbln1) as a novel cue that controls diverse aspects of axon growth and guidance throughout the central nervous system (CNS) by experiments using mouse and chick embryos. Cbln1 has previously been shown to function in late neural development to influence synapse organization. Here, we find that Cbln1 has an essential role in early neural development. Cbln1 is expressed on the axons and growth cones of developing commissural neurons and functions in an autocrine manner to promote axon growth. Cbln1 is also expressed in intermediate target tissues and functions as an attractive guidance cue. We find that these functions of Cbln1 are mediated by neurexin-2 (Nrxn2), which functions as the Cbln1 receptor for axon growth and guidance. In addition to the developing spinal cord, we further show that Cbln1 functions in diverse parts of the CNS with major roles in cerebellar parallel fiber growth and retinal ganglion cell axon guidance. Despite the prevailing role of Cbln1 as a synaptic organizer, our study discovers a new and unexpected function for Cbln1 as a general axon growth and guidance cue throughout the nervous system.


Assuntos
Axônios , Cerebelo , Embrião de Galinha , Animais , Camundongos , Axônios/metabolismo , Cerebelo/metabolismo , Medula Espinal/metabolismo , Neurônios/metabolismo , Proteínas do Tecido Nervoso/genética , Precursores de Proteínas/metabolismo
19.
Genes (Basel) ; 13(11)2022 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-36360284

RESUMO

Chickens are excellent models for the study of retinal development and function. Gene expression at the correct time is crucial to retinal development and function. The present study aimed to investigate retinal gene expression and morphology in locally grown chickens at various developmental stages. RNA was extracted from the retina at the embryonic and post-hatch stages, and the retinal layers were stained with haematoxylin and eosin (H&E). RT-PCR and RT-qPCR were used for gene expression analysis of 14 selected genes. The results showed that all the retinal genes were expressed at different developmental stages. However, there were slight noticeable variations in expression patterns. At the morphological level, all retinal layers were well observed, except for the outer plexiform layer that became visible in the fifteen-day chick embryo. The current study provides a baseline for standard retinal gene expression of 14 genes and retinal histological staining. The selected genes have different roles in retinal development and function, and most of these genes are associated with retinal diseases. The results obtained here can be applied to molecular retinal research and retinal diseases with genetic factors in retina animal models or human diseases.


Assuntos
Galinhas , Doenças Retinianas , Humanos , Embrião de Galinha , Animais , Galinhas/genética , Regulação da Expressão Gênica no Desenvolvimento , Retina/metabolismo , Doenças Retinianas/metabolismo , Expressão Gênica
20.
Nature ; 611(7935): 365-373, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36323783

RESUMO

Cells respond to physical stimuli, such as stiffness1, fluid shear stress2 and hydraulic pressure3,4. Extracellular fluid viscosity is a key physical cue that varies under physiological and pathological conditions, such as cancer5. However, its influence on cancer biology and the mechanism by which cells sense and respond to changes in viscosity are unknown. Here we demonstrate that elevated viscosity counterintuitively increases the motility of various cell types on two-dimensional surfaces and in confinement, and increases cell dissemination from three-dimensional tumour spheroids. Increased mechanical loading imposed by elevated viscosity induces an actin-related protein 2/3 (ARP2/3)-complex-dependent dense actin network, which enhances Na+/H+ exchanger 1 (NHE1) polarization through its actin-binding partner ezrin. NHE1 promotes cell swelling and increased membrane tension, which, in turn, activates transient receptor potential cation vanilloid 4 (TRPV4) and mediates calcium influx, leading to increased RHOA-dependent cell contractility. The coordinated action of actin remodelling/dynamics, NHE1-mediated swelling and RHOA-based contractility facilitates enhanced motility at elevated viscosities. Breast cancer cells pre-exposed to elevated viscosity acquire TRPV4-dependent mechanical memory through transcriptional control of the Hippo pathway, leading to increased migration in zebrafish, extravasation in chick embryos and lung colonization in mice. Cumulatively, extracellular viscosity is a physical cue that regulates both short- and long-term cellular processes with pathophysiological relevance to cancer biology.


Assuntos
Movimento Celular , Líquido Extracelular , Metástase Neoplásica , Neoplasias , Viscosidade , Animais , Embrião de Galinha , Camundongos , Actinas/metabolismo , Líquido Extracelular/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Trocadores de Sódio-Hidrogênio/metabolismo , Canais de Cátion TRPV , Peixe-Zebra/metabolismo , Metástase Neoplásica/patologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Via de Sinalização Hippo , Esferoides Celulares/patologia , Complexo 2-3 de Proteínas Relacionadas à Actina , Proteína rhoA de Ligação ao GTP , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Pulmão/patologia
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